Biofoundries provide an integrated infrastructure to enable the rapid design, construction, and testing of genetically reprogrammed organisms for biotechnology applications and research. Many biofoundries are being built and a Global Biofoundry Alliance has recently been established to coordinate activities worldwide.

INTRODUCTION: The Saccharomyces cerevisiae 2.0 project (Sc2.0) aims to modify the yeast genome with a series of densely spaced designer changes. Both a synthetic yeast chromosome arm (synIXR) and the entirely synthetic chromosome (synIII) function with high fitness in yeast. For designer genome synthesis projects, precise engineering of the physical sequence to match the specified design is important for the systematic evaluation of underlying design principles. Yeast can maintain nuclear chromosomes as rings, occurring by chance at repeated sequences, although the cyclized format is unfavorable in meiosis given the possibility of dicentric chromosome formation from meiotic recombination. Here, we describe the de novo synthesis of synthetic yeast chromosome V (synV) in the “Build-A-Genome China” course, perfectly matching the designer sequence and bearing loxPsym sites, distinguishable watermarks, and all the other features of the synthetic genome. We generated a ring synV derivative with user-specified cyclization coordinates and characterized its performance in mitosis and meiosis. RATIONALE: Systematic evaluation of underlying Sc2.0 design principles requires that the final assembled synthetic genome perfectly match the designed sequence. Given the size of yeast chromosomes, synthetic chromosome construction is performed iteratively, and new mutations and unpredictable events may occur during synthesis; even a very small number of unintentional nucleotide changes across the genome could have substantial effects on phenotype. Therefore, precisely matching the physical sequence to the designed sequence is crucial for verification of the design principles in genome synthesis. Ring chromosomes can extend those design principles to provide a model for genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders. RESULTS: We chemically synthesized, assembled, and incorporated designer chromosome synV (536,024 base pairs) of S. cerevisiae according to Sc2.0 principles, based on the complete nucleotide sequence of native yeast chromosome V (576,874 base pairs). This work was performed as part of the “Build-A-Genome China” course in Tianjin University. We corrected all mutations found—including duplications, substitutions, and indels—in the initial synV strain by using integrative cotransformation of the precise desired changes and by means of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)–based method. Altogether, 3331 corrected base pairs were required to match to the designed sequence. We generated a strain that exactly matches all designer sequence changes that displays high fitness under a variety of culture conditions. All corrections were verified with whole-genome sequencing; RNA sequencing revealed only minor changes in gene expression—most notably, decreases in expression of genes relocated near synthetic telomeres as a result of design. We constructed a functional circular synV (ring_synV) derivative in yeast by precisely joining both chromosome ends (telomeres) at specified coordinates. The ring chromosome showed restoration of subtelomeric gene expression levels. The ring_synV strain exhibited fitness comparable with that of the linear synV strain, revealed no change in sporulation frequency, but notably reduced spore viability. In meiosis, heterozygous or homozygous diploid ring_wtV and ring_synV chromosomes behaved similarly, exhibiting substantially higher frequency of the formation of zero-spore tetrads, a type that was not seen in the rod chromosome diploids. Rod synV chromosomes went through meiosis with high spore viability, despite no effort having been made to preserve meiotic competency in the design of synV. CONCLUSION: The perfect designer-matched synthetic chromosome V provides strategies to edit sequence variants and correct unpredictable events, such as off-target integration of extra copies of synthetic DNA elsewhere in the genome. We also constructed a ring synthetic chromosome derivative and evaluated its fitness and stability in yeast. Both synV and synVI can be circularized and can power yeast cell growth without affecting fitness when gene content is maintained. These fitness and stability phenotypes of the ring synthetic chromosome in yeast provide a model system with which to probe the mechanism of human ring chromosome disorders. Synthesis, cyclization, and characterization of synV .: ( A ) Synthetic chromosome V (synV, 536,024 base pairs) was designed in silico from native chromosome V (wtV, 576,874 base pairs), with extensive genotype modification designed to be phenotypically neutral. ( B ) CRISPR/Cas9 strategy for multiplex repair. ( C ) Colonies of wtV, synV, and ring_synV strains.

Synthetic biology is an emerging research field that focuses on using rational engineering strategies to program biological systems, conferring on them new functions and behaviours. By developing genetic parts and devices based on transcriptional, translational, post-translational modules, many genetic circuits and metabolic pathways had been programmed in single cells. Extending engineering capabilities from single-cell behaviours to multicellular microbial consortia represents a new frontier of synthetic biology. Herein, we first reviewed binary interaction modes of microorganisms in microbial consortia and their underlying molecular mechanisms, which lay the foundation of programming cell–cell interactions in synthetic microbial consortia. Systems biology studies on cellular systems enable systematic understanding of diverse physiological processes of cells and their interactions, which in turn offer insights into the optimal design of synthetic consortia. Based on such fundamental understanding, a comprehensive array of synthetic microbial consortia constructed in the last decade were reviewed, including isogenic microbial communities programmed by quorum sensing-based cell–cell communications, sender–receiver microbial communities with one-way communications, and microbial ecosystems wired by two-way (bi-directional) communications. Furthermore, many applications including using synthetic microbial consortia for distributed bio-computations, chemicals and bioenergy production, medicine and human health, and environments were reviewed. Synergistic development of systems and synthetic biology will provide both a thorough understanding of naturally occurring microbial consortia and rational engineering of these complicated consortia for novel applications.

Lignin-derived phenolic compounds are universal in the hydrolysate of pretreated lignocellulosic biomass. The phenolics reduce the efficiency of enzymatic hydrolysis and increase the cost of ethanol production. We investigated inhibition of phenolics on cellulase during enzymatic hydrolysis using vanillin as one of the typical lignin-derived phenolics and Avicel as cellulose substrate. As vanillin concentration increased from 0 to 10 mg/mL, cellulose conversion after 72-h enzymatic hydrolysis decreased from 53 to 26 %. Enzyme deactivation and precipitation were detected with the vanillin addition. The enzyme concentration and activity consecutively decreased during hydrolysis, but the inhibition degree, expressed as the ratio of the cellulose conversion without vanillin to the conversion with vanillin (A 0 /A), was almost independent on hydrolysis time. Inhibition can be mitigated by increasing cellulose loading or cellulase concentration. The inhibition degree showed linear relationship with the vanillin concentration and exponential relationship with the cellulose loading and the cellulase concentration. The addition of calcium chloride, BSA, and Tween 80 did not release the inhibition of vanillin significantly. pH and temperature for hydrolysis also showed no significant impact on inhibition degree. The presence of hydroxyl group, carbonyl group, and methoxy group in phenolics affected the inhibition degree. Besides phenolics concentration, other factors such as cellulose loading, enzyme concentration, and phenolic structure also affect the inhibition of cellulose conversion. Lignin-blocking agents have little effect on the inhibition effect of soluble phenolics, indicating that the inhibition mechanism of phenolics to enzyme is likely different from insoluble lignin. The inhibition of soluble phenolics can hardly be entirely removed by increasing enzyme concentration or adding blocking proteins due to the dispersity and multiple binding sites of phenolics than insoluble lignin.

INTRODUCTION: Design and construction of an extensively modified yeast genome is a direct means to interrogate the integrity, comprehensiveness, and accuracy of the knowledge amassed by the yeast community to date. The international synthetic yeast genome project (Sc2.0) aims to build an entirely designer, synthetic Saccharomyces cerevisiae genome. The synthetic genome is designed to increase genome stability and genetic flexibility while maintaining cell fitness near that of the wild type. A major challenge for a genome synthesis lies in identifying and eliminating fitness-reducing sequence variants referred to as “bugs.” RATIONALE: Debugging is imperative for successfully building a fit strain encoding a synthetic genome. However, it is time-consuming and laborious to replace wild-type genes and measure strain fitness systematically. The Sc2.0 PCRTag system, which specifies recoded sequences within open reading frames (ORFs), is designed to distinguish synthetic from wild-type DNA in a simple polymerase chain reaction (PCR) assay. This system provides an opportunity to efficiently map bugs to the related genes by using a pooling strategy and subsequently correct them. Further, as we identify bugs in designer sequences, we will identify gaps in our knowledge and gain a deeper understanding of genome biology, allowing refinement of future design strategies. RESULTS: We chemically synthesized yeast chromosome X, synX, designed to be 707,459 base pairs. A high-throughput mapping strategy called pooled PCRTag mapping (PoPM) was developed to identify unexpected bugs during chromosome assembly. With this method, the genotypes of pools of colonies with normal or defective fitness are assessed by PCRTag analysis. The PoPM method exploits the patchwork structure of synthetic and wild-type sequences observed in the majority of putative synthetic DNA integrants or meiotic progeny derived from synthetic/wild-type strain backcross. PCRTag analysis with both synthetic and wild-type specific primers, carried out with genomic DNA extracted from the two pools of clones (normal fitness versus a specific growth defect), can be used to identify regions of synthetic DNA missing from the normal fitness pool and, analogously, sections of wild-type DNA absent from the specific growth-defect pool. In this way, the defect can be efficiently mapped to a very small overlapping region, and subsequent systematic analysis of designed changes in that region can be used to identify the bug. Several bugs were identified and corrected, including a growth defect mapping to a specific synonymously recoded PCRTag sequence in the essential FIP1 ORF and the effect of introducing a loxPsym site that unexpectedly altered the the promoter function of a nearby gene, ATP2. In addition, meiotic crossover was employed to repair the massive duplications and rearrangements in the synthetic chromosome. The debugged synX strain exhibited high fitness under a variety of conditions tested and in competitive growth with the wild-type strain. CONCLUSION: Synthetic yeast chromosome X was chemically synthesized from scratch, a rigorous, incremental step toward complete synthesis of the whole yeast genome. Thousands of designer modifications in synX revealed extensive flexibility of the yeast genome. We developed an efficient mapping method, PoPM, to identify bugs during genome synthesis, generalizable to any watermarked synthetic chromosome, and several details of yeast biology were uncovered by debugging. Considering the numerous gene-associated PCRTags available in the synthetic chromosomes, PoPM may represent a powerful tool to map interesting phenotypes of mutated synthetic strains or even mutated wild-type strains to the relevant genes. It may also be useful to study yeast genetic interactions when an unexpected phenotype is generated by alterations in two or more genes, substantially expanding understanding of yeast genomic and cellular functions. The PoPM method is also likely to be useful for mapping phenotype(s) resulting from the genome SCRaMbLE system. Characterization of synX and debugging by pooled PCRTag mapping.: ( Top ) Design overview of synthetic chromosome X. ( Bottom ) Flow diagram of pooled PCRTag mapping (PoPM).

INTRODUCTION: It has long been an interesting question whether a living cell can be constructed from scratch in the lab, a goal that may not be realized anytime soon. Nonetheless, with advances in DNA synthesis technology, the complete genetic material of an organism can now be synthesized chemically. Hitherto, genomes of several organisms including viruses, phages, and bacteria have been designed and constructed. These synthetic genomes are able to direct all normal biological functions, capable of self-replication and production of offspring. Several years ago, a group of scientists worldwide formed an international consortium to reconstruct the genome of budding yeast, Saccharomyces cerevisiae . RATIONALE: The synthetic yeast genome, designated Sc2.0, was designed according to a set of arbitrary rules, including the elimination of transposable elements and incorporation of specific DNA elements to facilitate further genome manipulation. Among the 16 S. cerevisiae chromosomes, chromosome XII is unique as one of the longest yeast chromosomes (~1 million base pairs) and additionally encodes the highly repetitive ribosomal DNA locus, which forms the well-organized nucleolus. We report on the design, construction, and characterization of chromosome XII, the physically largest chromosome in S. cerevisiae. RESULTS: A 976,067–base pair linear chromosome, synXII, was designed based on the native chromosome XII sequence of S. cerevisiae , and chemically synthesized. SynXII was assembled using a two-step method involving, successive megachunk integration to produce six semisynthetic strains, followed by meiotic recombination–mediated assembly, yielding a full-length functional chromosome in S. cerevisiae. Minor growth defect “bugs” detected in synXII were caused by deletion of tRNA genes and were corrected by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit. The same synthetic rDNA unit was also used to regenerate rDNA at three distinct chromosomal locations. The rDNA signature sequences of the internal transcribed spacer (ITS), often used to determine species identity by standard DNA barcoding procedures, were swapped to generate a Saccharomyces synXII strain that would be identified as S. bayanus. Remarkably, these substantial DNA changes had no detectable phenotypic consequences under various laboratory conditions. CONCLUSION: The rDNA locus of synXII is highly plastic; not only can it be moved to other chromosomal loci, it can also be altered in its ITS region to masquerade as a distinct species as defined by DNA barcoding, used widely in taxonomy. The ability to perform “species morphing” reported here presumably reflects the degree of evolutionary flexibility by which these ITS regions change. However, this barcoding region is clearly not infinitely flexible, as only relatively modest intragenus base changes were tolerated. More severe intergenus differences in ITS sequence did not result in functional rDNAs, probably because of defects in rRNA processing. The ability to design, build, and debug a megabase-sized chromosome, together with the flexibility in rDNA locus position, speaks to the remarkable overall flexibility of the yeast genome. Hierarchical assembly and subsequent restructuring of synXII.: SynXII was assembled in two steps: First, six semisynthetic synXII strains were built in which segments of native XII DNA were replaced with the corresponding designer sequences. Next, the semisynthetic strains were combined withmultiple rounds ofmating/sporulation, eventually generating a single strain encoding fulllength synXII.The rDNA repeats were removed, modified, and subsequently regenerated at distinct chromosomal locations for species morphing and genome restructuring.