RuBisCO activity assays: a simplified biochemical redox approach for in vitro quantification and an RNA sensor approach for in vivo monitoring.

BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant soluble protein in nature. Extensive studies have been conducted for improving its activity in photosynthesis through approaches like protein engineering. Concurrently, multiple biochemical and radiolabeling assays have been developed for determining its activity. Although these existing assays yield reliable results, they require addition of multiple external components, rendering them less convenient and expensive. Therefore, in this study, we have developed two relatively cheaper, convenient, and easily reproducible assays for quantitative and qualitative estimation of RuBisCO activity. RESULTS: We simplified a contemporary NADH based spectrophotometric RuBisCO assay by using cyanobacterial cell lysate as the source for Calvin cycle enzymes. We analyzed the influence of inorganic carbon substrates, CO2 and NaHCO3, and varying protein concentrations on RuBisCO activity. Ribulose-1,5-bisphosphate (RuBP) consumption rates for the cultures grown under 5% CO2 were 5-7 times higher than the ones grown with 20 mM NaHCO3, at different protein concentrations. The difference could be due to the impaired activity of carbonic anhydrase in the cell lysate, which is required for the conversion of HCO3- to CO2. The highest RuBisCO activity of 2.13 nmol of NAD+/ µg of Chl-a/ min was observed with 50 µg of protein and 5% CO2. Additionally, we developed a novel RNA-sensor based fluorescence assay that is based on the principle of tracking the kinetics of ATP hydrolysis to ADP during the conversion of 3-phosphoglycerate (3-PG) to 1,3-bisphosphoglycerate (1,3-BPG) in the Calvin cycle. Under in vitro conditions, the fluorometric assay exhibited  ~ 3.4-fold slower reaction rate (0.37 min-1) than the biochemical assay when using 5% CO2. We also confirmed the in vivo application of this assay, where increase in the fluorescence was observed with the recombinant strain of Synechocystis sp. PCC 6803 (SSL142) expressing the ADP-specific RNA sensor, compared to the WT. In addition, SSL142 exhibited three-fold higher fluorescence when supplemented with 20 mM NaHCO3 as compared to the cells that were grown without NaHCO3 supplementation. CONCLUSIONS: Overall, we have developed a simplified biochemical assay for monitoring RuBisCO activity and demonstrated that it can provide reliable results as compared to the prior literature. Furthermore, the biochemical assay using 5% CO2 (100% relative activity) provided faster RuBP consumption rate compared to the biochemical assay utilizing 20 mM NaHCO3 (30.70% relative activity) and the in vitro fluorometric assay using 5% CO2 (29.64% relative activity). Therefore, the absorbance-based biochemical assay using 5% CO2 or higher would be suitable for in vitro quantification of the RuBisCO activity. On the other hand, the RNA-sensor based in vivo fluorometric assay can be applied for qualitative analysis and be used for high-throughput screening of RuBisCO variants. As RuBisCO is an enzyme shared amongst all the photoautotrophs, the assays developed in this study can easily be extended for analyzing the RuBisCO activities even in microalgae and higher plants.

Synergistic co-utilization of biomass-derived sugars enhances aromatic amino acid production by engineered Escherichia coli.

Efficient co-utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose-xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co-utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose-to-glucose flux ratio. A mutant copy of the xylose-specific activator (XylR) was then introduced into the phenylalanine-overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose-xylose co-utilization. Carbon contribution analysis through 13 C-fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose-xylose mixture; a threefold increase over NST74. Then, using biomass-derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9-fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L-h). This study presents a novel strategy for enhancing phenylalanine production through the co-utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products.

Unravelling the hidden power of esterases for biomanufacturing of short-chain esters.

Microbial production of esters has recently garnered wide attention, but the current production metrics are low. Evidently, the ester precursors (organic acids and alcohols) can be accumulated at higher titers by microbes like Escherichia coli. Hence, we hypothesized that their 'direct esterification' using esterases will be efficient. We engineered esterases from various microorganisms into E. coli, along with overexpression of ethanol and lactate pathway genes. High cell density fermentation exhibited the strains possessing esterase-A (SSL76) and carbohydrate esterase (SSL74) as the potent candidates. Fed-batch fermentation at pH 7 resulted in 80 mg/L of ethyl acetate and 10 mg/L of ethyl lactate accumulation by SSL76. At pH 6, the total ester titer improved by 2.5-fold, with SSL76 producing 225 mg/L of ethyl acetate, and 18.2 mg/L of ethyl lactate, the highest reported titer in E. coli. To our knowledge, this is the first successful demonstration of short-chain ester production by engineering 'esterases' in E. coli.

One cell-two wells bio-refinery: Demonstrating cyanobacterial chassis for co-production of heterologous and natural hydrocarbons.

In order to improve the potential of cyanobacterial cell factories, Synechococcus sp. PCC7002 was engineered as 'one cell-two wells bio-refinery', for ethylene ('heterologous' hydrocarbon) and carotenoids ('natural' metabolites) production, and demonstrating its outdoor performance. Although the cultures showed better production outdoor, they experienced multiple collapses during scale-up. Hence, flux balance analysis was performed which predicted higher ethylene production with increase in carbon input under outdoor light conditions. Furthermore, FBA predicted that ethylene production will not increase beyond a threshold carbon input flux, owing to limitations on ribulose-1,5-bisphosphate regeneration. Hence, a bicarbonate-supplementation strategy was devised. Cultures grown outdoor at optimal bicarbonate concentration (20 g/L) resulted in improved growth (0.141/h) and ethylene productivity (1.88 mL/L.h) for > 10 days, with enhanced carotenoid titres (40.4 mg/L). In a 100 L air-lift photo-bioreactor; cultures exhibited efficient ethylene (2.464 mL/L.h) and biomass (0.3 g/L.d) productivities, and carotenoids titres (64.4 mg/L), establishing a significant step towards commercialization.

Corynebacterium glutamicum as an Efficient Omnivorous Microbial Host for the Bioconversion of Lignocellulosic Biomass.

Corynebacterium glutamicum has been successfully employed for the industrial production of amino acids and other bioproducts, partially due to its native ability to utilize a wide range of carbon substrates. We demonstrated C. glutamicum as an efficient microbial host for utilizing diverse carbon substrates present in biomass hydrolysates, such as glucose, arabinose, and xylose, in addition to its natural ability to assimilate lignin-derived aromatics. As a case study to demonstrate its bioproduction capabilities, L-lactate was chosen as the primary fermentation end product along with acetate and succinate. C. glutamicum was found to grow well in different aromatics (benzoic acid, cinnamic acid, vanillic acid, and p-coumaric acid) up to a concentration of 40 mM. Besides, 13C-fingerprinting confirmed that carbon from aromatics enter the primary metabolism via TCA cycle confirming the presence of ß-ketoadipate pathway in C. glutamicum. 13C-fingerprinting in the presence of both glucose and aromatics also revealed coumarate to be the most preferred aromatic by C. glutamicum contributing 74 and 59% of its carbon for the synthesis of glutamate and aspartate respectively. 13C-fingerprinting also confirmed the activity of ortho-cleavage pathway, anaplerotic pathway, and cataplerotic pathways. Finally, the engineered C. glutamicum strain grew well in biomass hydrolysate containing pentose and hexose sugars and produced L-lactate at a concentration of 47.9 g/L and a yield of 0.639 g/g from sugars with simultaneous utilization of aromatics. Succinate and acetate co-products were produced at concentrations of 8.9 g/L and 3.2 g/L, respectively. Our findings open the door to valorize all the major carbon components of biomass hydrolysate by using C. glutamicum as a microbial host for biomanufacturing.

Corrigendum: Corynebacterium glutamicum as an efficient omnivorous microbial host for the bioconversion of lignocellulosic biomass.

[This corrects the article DOI: 10.3389/fbioe.2022.827386.].

A coculture-coproduction system designed for enhanced carbon conservation through inter-strain CO2 recycling.

Carbon loss in the form of CO2 is an intrinsic and persistent challenge faced during conventional and advanced biofuel production from biomass feedstocks. Current mechanisms for increasing carbon conservation typically require the provision of reduced co-substrates as additional reducing equivalents. This need can be circumvented, however, by exploiting the natural heterogeneity of lignocellulosic sugars mixtures and strategically using specific fractions to drive complementary CO2 emitting vs. CO2 fixing pathways. As a demonstration of concept, a coculture-coproduction system was developed by pairing two catabolically orthogonal Escherichia coli strains; one converting glucose to ethanol (G2E) and the other xylose to succinate (X2S). 13C-labeling studies reveled that G2E + X2S cocultures were capable of recycling 24% of all evolved CO2 and achieved a carbon conservation efficiency of 77%; significantly higher than the 64% achieved when all sugars are instead converted to just ethanol. In addition to CO2 exchange, the latent exchange of pyruvate between strains was discovered, along with significant carbon rearrangement within X2S.

Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction.

Ultra-low temperature (ULT) storage of microbial biomass is routinely practiced in biological laboratories. However, there is very little insight regarding the effects of biomass storage at ULT and the structure of the cell envelope, on cell viability. Eventually, these aspects influence bacterial cell lysis which is one of the critical steps for biomolecular extraction, especially protein extraction. Therefore, we studied the effects of ULT-storage (-80°C) on three different bacterial platforms: Escherichia coli, Bacillus subtilis and the cyanobacterium Synechocystis sp. PCC 6803. By using a propidium iodide assay and a modified MTT assay we determined the impact of ULT storage on cellular viability. Subsequently, the protein extraction efficiency was determined by analyzing the amount of protein released following the storage. The results successfully established that longer the ULT-storage time lower is the cell viability and larger is the protein extraction efficiency. Interestingly, E. coli and B. subtilis exhibited significant reduction in cell viability over Synechocystis 6803. This indicates that the cell membrane structure and composition may play a major role on cell viability in ULT storage. Interestingly, E. coli exhibited concomitant increase in cell lysis efficiency resulting in a 4.5-fold increase (from 109 µg/ml of protein on day 0 to 464 µg/ml of protein on day 2) in the extracted protein titer following ULT storage. Furthermore, our investigations confirmed that the protein function, tested through the extraction of fluorescent proteins from cells stored at ULT, remained unaltered. These results established the plausibility of using ULT storage to improve protein extraction efficiency. Towards this, the impact of shorter ULT storage time was investigated to make the strategy more time efficient to be adopted into protocols. Interestingly, E. coli transformants expressing mCherry yielded 2.7-fold increase (93 µg/mL to 254 µg/mL) after 10 mins, while 4-fold increase (380 µg/mL) after 120 mins of ULT storage in the extracted soluble protein. We thereby substantiate that: (1) the storage time of bacterial cells in -80°C affect cell viability and can alter protein extraction efficiency; and (2) exercising a simple ULT-storage prior to bacterial cell lysis can improve the desired protein yield without impacting its function.

High-throughput screening for efficient microbial biotechnology.

Microbial systems have been widely studied and exploited through genetic engineering to address industrial needs and societal challenges. However, owing to their complexity, singular approaches often do not yield desired or optimal results, pushing researchers to explore combinatorial strategies. With advances in synthetic biology, various methods can readily be employed to generate large and comprehensive libraries. To serve as tractable tools, however, this capability necessitates the development of high-throughput screening (HTS) techniques to identify the best performing strain and/or those carrying the desired trait. Owing to their miniaturization, time efficiency, potential for automation, and so on, HTS enables comprehensive exploration of diverse experimental landscapes. Herein, we review the recent and novel HTS approaches and applications in the realm of microbial biotechnology.

Hybrid phenolic-inducible promoters towards construction of self-inducible systems for microbial lignin valorization.

BACKGROUND: Engineering strategies to create promoters that are both higher strength and tunable in the presence of inexpensive compounds are of high importance to develop metabolic engineering technologies that can be commercialized. Lignocellulosic biomass stands out as the most abundant renewable feedstock for the production of biofuels and chemicals. However, lignin a major polymeric component of the biomass is made up of aromatic units and remains as an untapped resource. Novel synthetic biology tools for the expression of heterologous proteins are critical for the effective engineering of a microbe to valorize lignin. This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics present in lignocellulosic hydrolysates to increase heterologous protein production. RESULTS: A hybrid promoter engineering approach was utilized for the construction of phenolic-inducible promoters of higher strength. The hybrid promoters were constructed by replacing the spacer region of an endogenous promoter, PemrR present in E. coli that was naturally inducible by phenolics. In the presence of vanillin, the engineered promoters Pvtac, Pvtrc, and Pvtic increased protein expression by 4.6-, 3.0-, and 1.5-fold, respectively, in comparison with a native promoter, PemrR. In the presence of vanillic acid, Pvtac, Pvtrc, and Pvtic improved protein expression by 9.5-, 6.8-, and 2.1-fold, respectively, in comparison with PemrR. Among the cells induced with vanillin, the emergence of a sub-population constituting the healthy and dividing cells using flow cytometry was observed. The analysis also revealed this smaller sub-population to be the primary contributor for the increased expression that was observed with the engineered promoters. CONCLUSIONS: This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics to increase heterologous protein production. Employing promoters inducible by phenolics will provide the following advantages: (1) develop substrate inducible systems; (2) lower operating costs by replacing expensive IPTG currently used for induction; (3) develop dynamic regulatory systems; and (4) provide flexibility in operating conditions. The flow cytometry findings strongly suggest the need for novel approaches to maintain a healthy cell population in the presence of phenolics to achieve increased heterologous protein expression and, thereby, valorize lignin efficiently.

Lignin Valorization: Two Hybrid Biochemical Routes for the Conversion of Polymeric Lignin into Value-added Chemicals.

Naturally, many aerobic organisms degrade lignin-derived aromatics through conserved intermediates including protocatechuate and catechol. Employing this microbial approach offers a potential solution for valorizing lignin into valuable chemicals for a potential lignocellulosic biorefinery and enabling bioeconomy. In this study, two hybrid biochemical routes combining lignin chemical depolymerization, plant metabolic engineering, and synthetic pathway reconstruction were demonstrated for valorizing lignin into value-added products. In the biochemical route 1, alkali lignin was chemically depolymerized into vanillin and syringate as major products, which were further bio-converted into cis, cis-muconic acid (ccMA) and pyrogallol, respectively, using engineered Escherichia coli strains. In the second biochemical route, the shikimate pathway of Tobacco plant was engineered to accumulate protocatechuate (PCA) as a soluble intermediate compound. The PCA extracted from the engineered Tobacco was further converted into ccMA using the engineered E. coli strain. This study reports a direct process for converting lignin into ccMA and pyrogallol as value-added chemicals, and more importantly demonstrates benign methods for valorization of polymeric lignin that is inherently heterogeneous and recalcitrant. Our approach also validates the promising combination of plant engineering with microbial chassis development for the production of value added and speciality chemicals.

Decoding how a soil bacterium extracts building blocks and metabolic energy from ligninolysis provides road map for lignin valorization.

Sphingobium sp. SYK-6 is a soil bacterium boasting a well-studied ligninolytic pathway and the potential for development into a microbial chassis for lignin valorization. An improved understanding of its metabolism will help researchers in the engineering of SYK-6 for the production of value-added chemicals through lignin valorization. We used 13C-fingerprinting, 13C metabolic flux analysis (13C-MFA), and RNA-sequencing differential expression analysis to uncover the following metabolic traits: (i) SYK-6 prefers alkaline conditions, making it an efficient host for the consolidated bioprocessing of lignin, and it also lacks the ability to metabolize sugars or organic acids; (ii) the CO2 release (i.e., carbon loss) from the ligninolysis-based metabolism of SYK-6 is significantly greater than the CO2 release from the sugar-based metabolism of Escherichia coli; (iii) the vanillin catabolic pathway (which is the converging point of majority of the lignin catabolic pathways) is coupled with the tetrahydrofolate-dependent C1 pathway that is essential for the biosynthesis of serine, histidine, and methionine; (iv) catabolic end products of lignin (pyruvate and oxaloacetate) must enter the tricarboxylic acid (TCA) cycle first and then use phosphoenolpyruvate carboxykinase to initiate gluconeogenesis; and (v) 13C-MFA together with RNA-sequencing differential expression analysis establishes the vanillin catabolic pathway as the major contributor of NAD(P)H synthesis. Therefore, the vanillin catabolic pathway is essential for SYK-6 to obtain sufficient reducing equivalents for its healthy growth; cosubstrate experiments support this finding. This unique energy feature of SYK-6 is particularly interesting because most heterotrophs rely on the transhydrogenase, the TCA cycle, and the oxidative pentose phosphate pathway to obtain NADPH.

Boosting D-lactate production in engineered cyanobacteria using sterilized anaerobic digestion effluents.

Anaerobic digestion (AD) is an environmentally friendly approach to waste treatment, which can generate N and P-rich effluents that can be used as nutrient sources for microalgal cultivations. Modifications of AD processes to inhibit methanogenesis leads to the accumulation of acetic acid, a carbon source that can promote microalgal biosynthesis. This study tested different AD effluents from municipal wastes on their effect on D-lactate production by an engineered Synechocystis sp. PCC 6803 (carrying a novel lactate dehydrogenase). The results indicate that: (1) AD effluents can be supplemented into the modified BG-11 culture medium (up to 1:4 volume ratio) to reduce N and P cost; (2) acetate-rich AD effluents enhance D-lactate synthesis by ∼ 40% (1.2g/L of D-lactate in 20 days); and (3) neutral or acidic medium had a deleterious effect on lactate secretion and biomass growth by the engineered strain. This study demonstrates the advantages and guidelines in employing wastewater for photomixotrophic biosynthesis using engineered microalgae.

Elucidation of intrinsic biosynthesis yields using 13C-based metabolism analysis.

This paper discusses the use of 13C-based metabolism analysis for the assessment of intrinsic product yields - the actual carbon contribution from a single carbon substrate to the final product via a specific biosynthesis route - in the following four cases. First, undefined nutrients (such as yeast extract) in fermentation may contribute significantly to product synthesis, which can be quantified through an isotopic dilution method. Second, product and biomass synthesis may be dependent on the co-metabolism of multiple-carbon sources. 13C labeling experiments can track the fate of each carbon substrate in the cell metabolism and identify which substrate plays a main role in product synthesis. Third, 13C labeling can validate and quantify the contribution of the engineered pathway (versus the native pathway) to the product synthesis. Fourth, the loss of catabolic energy due to cell maintenance (energy used for functions other than production of new cell components) and low P/O ratio (Phosphate/Oxygen Ratio) significantly reduces product yields. Therefore, 13C-metabolic flux analysis is needed to assess the influence of suboptimal energy metabolism on microbial productivity, and determine how ATP/NAD(P)H are partitioned among various cellular functions. Since product yield is a major determining factor in the commercialization of a microbial cell factory, we foresee that 13C-isotopic labeling experiments, even without performing extensive flux calculations, can play a valuable role in the development and verification of microbial cell factories.

Photoautotrophic production of D-lactic acid in an engineered cyanobacterium.

BACKGROUND: The world faces the challenge to develop sustainable technologies to replace thousands of products that have been generated from fossil fuels. Microbial cell factories serve as promising alternatives for the production of diverse commodity chemicals and biofuels from renewable resources. For example, polylactic acid (PLA) with its biodegradable properties is a sustainable, environmentally friendly alternative to polyethylene. At present, PLA microbial production is mainly dependent on food crops such as corn and sugarcane. Moreover, optically pure isomers of lactic acid are required for the production of PLA, where D-lactic acid controls the thermochemical and physical properties of PLA. Henceforth, production of D-lactic acid through a more sustainable source (CO2) is desirable. RESULTS: We have performed metabolic engineering on Synechocystis sp. PCC 6803 for the phototrophic synthesis of optically pure D-lactic acid from CO2. Synthesis of optically pure D-lactic acid was achieved by utilizing a recently discovered enzyme (i.e., a mutated glycerol dehydrogenase, GlyDH*). Significant improvements in D-lactic acid synthesis were achieved through codon optimization and by balancing the cofactor (NADH) availability through the heterologous expression of a soluble transhydrogenase. We have also discovered that addition of acetate to the cultures improved lactic acid production. More interestingly, (13)C-pathway analysis revealed that acetate was not used for the synthesis of lactic acid, but was mainly used for synthesis of certain biomass building blocks (such as leucine and glutamate). Finally, the optimal strain was able to accumulate 1.14 g/L (photoautotrophic condition) and 2.17 g/L (phototrophic condition with acetate) of D-lactate in 24 days. CONCLUSIONS: We have demonstrated the photoautotrophic production of D-lactic acid by engineering a cyanobacterium Synechocystis 6803. The engineered strain shows an excellent D-lactic acid productivity from CO2. In the late growth phase, the lactate production rate by the engineered strain reached a maximum of ~0.19 g D-lactate/L/day (in the presence of acetate). This study serves as a good complement to the recent metabolic engineering work done on Synechocystis 6803 for L-lactate production. Thereby, our study may facilitate future developments in the use of cyanobacterial cell factories for the commercial production of high quality PLA.

Metabolic engineering of Synechocystis sp. strain PCC 6803 for isobutanol production.

Global warming and decreasing fossil fuel reserves have prompted great interest in the synthesis of advanced biofuels from renewable resources. In an effort to address these concerns, we performed metabolic engineering of the cyanobacterium Synechocystis sp. strain PCC 6803 to develop a strain that can synthesize isobutanol under both autotrophic and mixotrophic conditions. With the expression of two heterologous genes from the Ehrlich pathway, the engineered strain can accumulate 90 mg/liter of isobutanol from 50 mM bicarbonate in a gas-tight shaking flask. The strain does not require any inducer (i.e., isopropyl ß-d-1-thiogalactopyranoside [IPTG]) or antibiotics to maintain its isobutanol production. In the presence of glucose, isobutanol synthesis is only moderately promoted (titer = 114 mg/liter). Based on isotopomer analysis, we found that, compared to the wild-type strain, the mutant significantly reduced its glucose utilization and mainly employed autotrophic metabolism for biomass growth and isobutanol production. Since isobutanol is toxic to the cells and may also be degraded photochemically by hydroxyl radicals during the cultivation process, we employed in situ removal of the isobutanol using oleyl alcohol as a solvent trap. This resulted in a final net concentration of 298 mg/liter of isobutanol under mixotrophic culture conditions.

Statistics-based model for prediction of chemical biosynthesis yield from Saccharomyces cerevisiae.

BACKGROUND: The robustness of Saccharomyces cerevisiae in facilitating industrial-scale production of ethanol extends its utilization as a platform to synthesize other metabolites. Metabolic engineering strategies, typically via pathway overexpression and deletion, continue to play a key role for optimizing the conversion efficiency of substrates into the desired products. However, chemical production titer or yield remains difficult to predict based on reaction stoichiometry and mass balance. We sampled a large space of data of chemical production from S. cerevisiae, and developed a statistics-based model to calculate production yield using input variables that represent the number of enzymatic steps in the key biosynthetic pathway of interest, metabolic modifications, cultivation modes, nutrition and oxygen availability. RESULTS: Based on the production data of about 40 chemicals produced from S. cerevisiae, metabolic engineering methods, nutrient supplementation, and fermentation conditions described therein, we generated mathematical models with numerical and categorical variables to predict production yield. Statistically, the models showed that: 1. Chemical production from central metabolic precursors decreased exponentially with increasing number of enzymatic steps for biosynthesis (>30% loss of yield per enzymatic step, P-value = 0); 2. Categorical variables of gene overexpression and knockout improved product yield by 2~4 folds (P-value < 0.1); 3. Addition of notable amount of intermediate precursors or nutrients improved product yield by over five folds (P-value < 0.05); 4. Performing the cultivation in a well-controlled bioreactor enhanced the yield of product by three folds (P-value < 0.05); 5. Contribution of oxygen to product yield was not statistically significant. Yield calculations for various chemicals using the linear model were in fairly good agreement with the experimental values. The model generally underestimated the ethanol production as compared to other chemicals, which supported the notion that the metabolism of Saccharomyces cerevisiae has historically evolved for robust alcohol fermentation. CONCLUSIONS: We generated simple mathematical models for first-order approximation of chemical production yield from S. cerevisiae. These linear models provide empirical insights to the effects of strain engineering and cultivation conditions toward biosynthetic efficiency. These models may not only provide guidelines for metabolic engineers to synthesize desired products, but also be useful to compare the biosynthesis performance among different research papers.

Evaluating factors that influence microbial synthesis yields by linear regression with numerical and ordinal variables.

In the production of chemicals via microbial fermentation, achieving a high yield is one of the most important objectives. We developed a statistical model to analyze influential factors that determine product yield by compiling data obtained from engineered Escherichia coli developed within last 10 years. Using both numerical and ordinal variables (e.g., enzymatic steps, cultivation conditions, and genetic modifications) as input parameters, our model revealed that cultivation modes, nutrient supplementation, and oxygen conditions were the three significant factors for improving product yield. Generally, the model showed that product yield decreases as the number of enzymatic steps in the biosynthesis pathway increases (7-9% loss of yield per enzymatic step). Moreover, overexpression of enzymes or removal of competitive pathways (e.g., knockout) does not necessarily result in an amplification of product yield (P-value>0.1), possibly because of limited capacity in the biosynthesis pathway to accommodate an increase in flux. The model not only provides general guidelines for metabolic engineering and fermentation processes, but also allows a priori estimation and comparison of product yields under designed cultivation conditions.