Anshu Shankar Mathur

Metabolic engineering and synthetic biology are enhancing the design and production of microbial cells with scaled capabilities for producing high-demand bio-based products, such as biofuels, enzymes, nutraceuticals, and food industry products. Metabolic engineering provides cellular metabolism to maximize product yields and minimize wastages. Synthetic biology further modifies and enables the production of complex molecules that are difficult to synthesize through other methods. This chapter provides an in-depth exploration and study of the latest innovations and developments, highlighting their transformative impact on optimized biomanufacturing processes. It also highlights the advanced genetic tools, genome-scale modelling, etc. Additionally, it discusses prospects, challenges, and potential solutions for next-generation biomanufacturing, emphasizing the role of Artificial Intelligence (AI) and Machine Learning (ML) in system metabolic engineering.

A novel approach for in situ transesterification, extraction, separation, and purification of fatty acid ethyl esters (FAEE) for biodiesel and docosahexaenoic acid (DHA) from Thraustochytrid biomass has been developed. The downstream processing of Thraustochytrids oil necessitates optimization, considering the higher content of polyunsaturated fatty acids (PUFA). While two‐step methods are commonly employed for extracting and transesterifying oil from oleaginous microbes, this may result in oxidation/epoxidation of omega‐3 oil due to prolonged exposure to heat and oxygen. To address this issue, a rapid single‐step method was devised for in situ transesterification of Thraustochytrid oil. Through further process optimization, a 50% reduction in solvent requirement was achieved without significantly impacting fatty acid recovery or composition. Scale‐up studies in a 4 L reactor demonstrated complete FAEE recovery (99.98% of total oil) from biomass, concurrently enhancing DHA yield from 16% to nearly 22%. The decolorization of FAEE oil with fuller's earth effectively removed impurities such as pigments, secondary metabolites, and waxes, resulting in a clear, shiny appearance. High‐performance liquid chromatography (HPLC) analysis indicated that the eluted DHA was over 94.5% pure, as corroborated by GC‐FID analysis.

The study focuses on the simultaneous improvement of biomass, lipid, and docosahexaenoic acid (DHA) productivities in a single reactor using modulator cont

Metabolite production by filamentous fungi hampered because of high viscosity generated during growth. Low viscosity fermentation by mold is one of the preferred ways of large scale enzymes production. Cellulolytic enzymes play a key role during the process of lignocellulosic biomass conversion. In this study, a mutant RC‐23‐1 was isolated through mutagenesis (diethyl sulfate followed by UV) of Trichoderma reesei RUT‐C30. RCRC‐23‐1 not only gave higher cellulase production but also generated lower viscosity during enzyme production. Viscosity of mutant growth was more than three times lower than parent strain. RC‐23‐1 shows unique, yeast‐like colony morphology on solid media and small pellet‐like growth in liquid media. This mutant did not spread like mold on solid media. This mutant produces cellulases constitutively when grown in sugars. Using only glucose, the cellulase production was 4.1 FPU/ml. Among polysaccharides (avicel, xylan, and pectin), avicel gave maximum of 6.2 FPU/ml and pretreated biomass (rice straw, wheat straw and sugarcane bagasse) produced 5.1–5.8 FPU/ml. At 7 L scale reactor, fed‐batch process was designed for cellulase production using different carbon and nitrogen sources. Maximum yield of cellulases was 182 FPU/g of lactose consumed was observed in fed‐batch process. The produced enzyme used for hydrolysis of acid pretreated rice straw (20% solid loading) and maximum of 60% glucan conversion was observed. RC‐23‐1 mutant is good candidate for large scale cellulase production and could be a model strain to study mold to yeast‐like transformation.

The present study provides an integrated method for utilizing the wastewaters from second generation (2G) ethanol pretreatment plant for microalgal biomass and lipid production. The study was conducted using a mixture of wastewaters (referred as MW; pH 4.3) generated after washing of acidic and alkaline-soaked lignocellulosic biomass prior to pretreatment process. The growth studies indicated that the thermotolerant strain of Chlorella pyrenoidosa (C. pyrenoidosa) M18 exhibited higher cell proliferation in wastewater as compared to freshwater. About 20–25% enhancement in biomass (509 mg L⁻¹ d⁻¹ ± 3.09) and lipid productivity (146 mg L⁻¹ d⁻¹ ± 1.34) was observed in MW. The total chlorophyll content and variable fluorescence by maximum fluorescence (Fv/Fm) ratio of strain cultivated in MW were 10.32 µg mL⁻¹ and 0.75, respectively. The use of MW also enhanced the content of saturated and monounsaturated fatty acids in total lipid. The exhausted wastewater medium obtained after harvesting the auto-flocculated biomass was also reused up to three successive growth cycles. The recycled medium without any nutrient addition could be used for two subsequent rounds with enhanced biomass (520 mg L⁻¹ d⁻¹ ± 4.07) and lipid (157.71 mg L⁻¹ d⁻¹ ± 1.09) productivities. This synergistic approach of cultivating thermotolerant microalgae with wastewater from 2G pretreatment plant provides an economical setup for development of commercial algal biofuel technology.

Efforts to develop efficient lignocellulose-degrading enzymatic preparations have led to the relatively recent discovery of a new class of novel cellulase boosters, termed lytic polysaccharide monoxygenases (LPMOs). These enzymes are copper-dependent metalloenzymes that initiate the biomass deconstruction process and subsequently work together with cellulases, hemicellulases, and other accessory enzymes to enhance their hydrolytic action. Given their wide distribution and diversity, screening and isolation of potent LPMOs from natural fungal diversity may provide an important avenue for increasing the efficiency of cellulases and thereby decreasing cellulosic ethanol production costs. However, methods for quick screening and detection are still not widely available. In this article, a simple and sensitive method is described by combining nonhydrolytic activity enhancement followed by LC–MS-based quantitation of LPMOs. In this study, a screening approach has been developed for the detection of nonhydrolytic cellulase-enhancing enzymes in crude fungal supernatants. With the application of a saturating benchmark cocktail of Celluclast 1.5L, fungal isolates were selected which had the capability of hydrolyzing pretreated rice straw by their synergistic enzyme fractions. Subsequently, these fungal extracts along with an LPMO-enriched commercial enzyme were investigated for their ability to produce Type I LPMO activity. An LC–MS-based methodology was applied to quantitate gluconic acid in enzymatic hydrolysates as an indirect measurement of Type I LPMO activity. The present study describes an LC–MS-based separation method to detect and quantitate LPMO activity in a commercial enzyme. This method was also applied to screen fungal extracts. The developed screening strategy has enabled detection of LPMO activity in two industrially important Penicillium strains.

BACKGROUND: Efficient conversion of inhibitor containing pentose rich stream into bioethanol or biogas is still challenging due to several technological challenges; however, this stream can be efficiently assimilated into lipids by oleaginous yeasts such as Rhodotorula. In this work, a continuous and nonsterile process was developed for bioconversion of waste streams into lipids for biodiesel using growth‐inhibitor‐tolerant oleaginous yeast.RESULTS: An inhibitor‐tolerating property of the strain was used to develop a cyclic and continuous near axenic cultivation process in a 2 L nonsterile bioreactor to enhance valorization of waste streams and to reduce energy requirements for media and vessel sterilization. After media optimization, continuous cultivation resulted in significant increase in biomass (14.90 g L⁻¹ day⁻¹) and lipid productivity (6.15 g L⁻¹ day⁻¹) as compared to flask (biomass ∼3 g L⁻¹ day⁻¹ and lipid productivity ∼0.6 g L⁻¹ day⁻¹) results. Palmitic acid (C16:0) and oleic acid (C18:1) were the major fatty acids, accounting for almost 80% of total lipids, thus making lipid produced in the process suitable for biodiesel production.CONCLUSION: Inhibitors present in waste streams successfully maintained near axenic conditions during the cultivation. Maximum dilution rate of 1 was achieved after media optimization, which resulted in complete valorization of nutrients into biomass and lipids with high productivity. © 2018 Society of Chemical Industry