Understanding the metabolism of the microbial host is essential for the development and optimization of whole-cell based biocatalytic processes, as it dictates production efficiency. This is especially true for redox biocatalysis where metabolically active cells are employed because of the cofactor/cosubstrate regenerative capacity endogenous in the host. Recombinant Escherichia coli was used for overproducing proline-4-hydroxylase (P4H), a dioxygenase catalyzing the hydroxylation of free l-proline into trans-4-hydroxy-l-proline with a-ketoglutarate (a-KG) as cosubstrate. In this whole-cell biocatalyst, central carbon metabolism provides the required cosubstrate a-KG, coupling P4H biocatalytic performance directly to carbon metabolism and metabolic activity. By applying both experimental and computational biology tools, such as metabolic engineering and 13C-metabolic flux analysis (13C-MFA), we investigated and quantitatively described the physiological, metabolic, and bioenergetic response of the whole-cell biocatalyst to the targeted bioconversion and identified possible metabolic bottlenecks for further rational pathway engineering. A proline degradation-deficient E. coli strain was constructed by deleting the putA gene encoding proline dehydrogenase. Whole-cell biotransformations with this mutant strain led not only to quantitative proline hydroxylation but also to a doubling of the specific trans-4-l-hydroxyproline (hyp) formation rate, compared to the wild type. Analysis of carbon flux through central metabolism of the mutant strain revealed that the increased a-KG demand for P4H activity did not enhance the a-KG generating flux, indicating a tightly regulated TCA cycle operation under the conditions studied. In the wild type strain, P4H synthesis and catalysis caused a reduction in biomass yield. Interestingly, the ΔputA strain additionally compensated the associated ATP and NADH loss by reducing maintenance energy demands at comparably low glucose uptake rates, instead of increasing the TCA activity. The putA knockout in recombinant E. coli BL21(DE3)(pLysS) was found to be promising for productive P4H catalysis not only in terms of biotransformation yield, but also regarding the rates for biotransformation and proline uptake and the yield of hyp on the energy source. The results indicate that, upon a putA knockout, the coupling of the TCA-cycle to proline hydroxylation via the cosubstrate a-KG becomes a key factor constraining and a target to further improve the efficiency of a-KG-dependent biotransformations.

Flavonoids and terpenoids are promising candidates for use as functional foods and novel therapeutics due to their prominent biological activities. However, the use of several bioactive plant compounds is limited by low stability and solubility problems. This review underlines the feasibility of enzymatic synthesis of novel bioactive analogs of selected flavonoids (silybin, rutin) and terpenoids (perillyl alcohol, POH) in nonconventional reaction systems. The effect of various parameters on the enzymatic acylation and/or glucosylation of these phytochemicals was studied. Also, the role of the structure of the novel molecules in relation to their biological function was investigated in various cancer cell lines.

Cyanobacteria play an important role in photobiotechnology. Yet, one of their key central metabolic pathways, the tricarboxylic acid (TCA) cycle, has a unique architecture compared to most heterotrophs and still remains largely unexploited. The conversion of 2-oxoglutarate to succinate via succinyl-CoA is absent but is by-passed by several other reactions. Overall, fluxes under photoautotrophic growth conditions through the TCA cycle are low, which has implications for the production of chemicals. In this study, we investigate the capacity of the TCA cycle of Synechocystis sp PCC 6803 for the production of trans-4-hydroxy-L-proline (Hyp), a valuable chiral building block for the pharmaceutical and cosmetic industries. For the first time, photoautotrophic Hyp production was achieved in a cyanobacterium expressing the gene for the L-proline-4-hydroxylase (P4H) from Dactylosporangium sp. strain RH1. Interestingly, while elevated intracellular Hyp concentrations could be detected in the recombinant Synechocystis strains under all tested conditions, detectable Hyp secretion into the medium was only observed when the pH of the medium exceeded 9.5 and mostly in the late phases of the cultivation. We compared the rates obtained for autotrophic Hyp production with published sugar-based production rates in E. coli. The land-use efficiency (space-time yield) of the phototrophic process is already in the same order of magnitude as the heterotrophic process considering sugar farming as well. But, the remarkable plasticity of the cyanobacterial TCA cycle promises the potential for a 23–55 fold increase in space-time yield when using Synechocystis. Altogether, these findings contribute to a better understanding of bioproduction from the TCA cycle in photoautotrophs and broaden the spectrum of chemicals produced in metabolically engineered cyanobacteria.

Yarrowia lipolytica is a non-pathogenic aerobic yeast with numerous industrial biotechnology applications. The organism grows in a wide variety of media, industrial byproducts, and wastes. A need exists for molecular tools to improve heterologous protein expression and pathway reconstitution. In an effort to identify strong native promoters in glycerol-based media, six highly expressed genes were mined from public data, analyzed, and validated. The promoters from the three most highly expressed (H3, ACBP, and TMAL) were cloned upstream of the reporter mCherry in episomal and integrative vectors. Fluorescence was quantified by flow cytometry and promoter strength was benchmarked with known strong promoters (pFBA1in, pEXP1, and pTEF1in) in cells growing in glucose, glycerol, and synthetic glycerol media. The results show that pH3 > pTMAL > pACBP are very strong promoters, with pH3 exceeding all other tested promoters. Hybrid promoters were also constructed, linking the Upstream Activating Sequence 1B (UAS1B8) with H3(260) or TMAL(250) minimal promoters, and compared to the UAS1B8-TEF1(136) promoter. The new hybrid promoters exhibited far superior strength. The novel promoters were utilized to overexpress the lipase LIP2, achieving very high secretion levels. In conclusion, our research identified and characterized several strong Y. lipolytica promoters that expand the capacity to engineer Yarrowia strains and valorize industrial byproducts.