Pichia pastoris is one of the most widely used host for the production of recombinant proteins. Expression systems that rely mostly on promoters from genes encoding alcohol oxidase 1 or glyceraldehyde-3-phosphate dehydrogenase have been developed together with related bioreactor operation strategies based on carbon sources such as methanol, glycerol, or glucose. Although, these processes are relatively efficient and easy to use, there have been notable improvements over the last twenty years to better control gene expression from these promoters and their engineered variants. Methanol-free and more efficient protein production platforms have been developed by engineering promoters and transcription factors. The production window of P. pastoris has been also extended by using alternative feedstocks including ethanol, lactic acid, mannitol, sorbitol, sucrose, xylose, gluconate, formate, or rhamnose. Herein, the specific aspects that are emerging as key parameters for recombinant protein synthesis are discussed. For this purpose, a holistic approach has been considered to scrutinize protein production processes from strain design to bioprocess optimization, particularly focusing on promoter engineering, transcriptional circuitry redesign. This review also considers the optimization of bioprocess based on alternative carbon sources and derived co-feeding strategies. Optimization strategies for recombinant protein synthesis through metabolic modelling are also discussed.

Yeast was the first microorganism used by mankind for biotransformation processes that laid the foundations of industrial biotechnology. In the last decade,Pichia pastorishas become the leading eukaryotic host organism for bioproduct generation. Most of theP. pastorisbioprocess operations has been relying on toxic methanol and glucose feed. In the actual bioeconomy era, for sustainable value-added bioproduct generation, non-conventional yeastP. pastorisbioprocess operations should be extended to low-cost and renewable substrates for large volume bio-based commodity productions. In this review, we evaluated the potential ofP. pastorisfor the establishment of circular bioeconomy due to its potential to generate industrially relevant bioproducts from renewable sources and waste streams in a cost-effective and environmentally friendly manner. Furthermore, we discussed challenges with the second generationP. pastorisplatforms and propose novel insights for future perspectives. In this regard, potential of low cost substrate candidates, i.e., lignocellulosic biomass components, cereal by-products, sugar industry by-products molasses and sugarcane bagasse, high fructose syrup by-products, biodiesel industry by-product crude glycerol, kitchen waste and other agri-food industry by products were evaluated forP. pastoriscell growth promoting effects and recombinant protein production. Further metabolic pathway engineering ofP. pastoristo construct renewable and low cost substrate utilization pathways was discussed. Although, second generationP. pastorisbioprocess operations for valorisation of wastes and by-products still in its infancy, rapidly emerging synthetic biology tools and metabolic engineering ofP. pastoriswill pave the way for more sustainable environment and bioeconomy. From environmental point of view, second generation bioprocess development is also important for waste recycling otherwise disposal of carbon-rich effluents creates environmental concerns.P. pastorishigh tolerance to toxic contaminants found in lignocellulosic biomass hydrolysate and industrial waste effluent crude glycerol provides the yeast with advantages to extend its applications toward second generationP. pastorisstrain design and bioprocess engineering, in the years to come.Graphical Abstract

In recent years, CO₂ reduction and utilization have been proposed as an innovative solution for global warming and the ever-growing energy and raw material demands. In contrast to various classical methods, including chemical, electrochemical, and photochemical methods, enzymatic methods offer a green and sustainable option for CO₂ conversion. In addition, enzymatic hydrogenation of CO₂ into platform chemicals could be used to produce economically useful hydrogen storage materials, making it a win-win strategy. The thermodynamic and kinetic stability of the CO₂ molecule makes its utilization a challenging task. However, Nicotine adenine dinucleotide (NAD⁺)-dependent formate dehydrogenases (FDHs), which have high selectivity and specificity, are attractive catalysts to overcome this issue and convert CO₂ into fuels and renewable chemicals. It is necessary to improve the stability, cofactor necessity, and CO₂ conversion efficiency of these enzymes, such as by combining them with appropriate hybrid systems. However, metal-independent, NAD⁺-dependent FDHs, and their CO₂ reduction activity have received limited attention to date. This review outlines the CO₂ reduction ability of these enzymes as well as their properties, reaction mechanisms, immobilization strategies, and integration with electrochemical and photochemical systems for the production of formic acid or formate. The biotechnological applications of FDH, future perspectives, barriers to CO₂ reduction with FDH, and aspects that must be further developed are briefly summarized. We propose that constructing hybrid systems that include NAD⁺-dependent FDHs is a promising approach to convert CO₂ and strengthen the sustainable carbon bio-economy.